Mechanism(s) by which ebolavirus enters cells, and the identity of the ebolavirus receptor(s) remain elusive. We have recently determined the crystal structure of Zaire ebolavirus GP in its trimeric, prefusion conformation (3 GP1 + 3 GP2). This is the first near complete structure of any filoviral glycoprotein and now provides the first images of the putative receptor binding site. We hypothesize that the receptor binding site (RBS) occupies a ~25 residue, conformational footprint of GP1. This site is hidden on native GP as it is sequestered in the bowl of the GP trimer and further masked by heavily glycosylated regions that project from the GP surface. Importantly, our crystal structure identifies the probable cathepsin cleavage site of GP and illustrates how the RBS would be unsheathed upon cathepsin cleavage. We propose a model of ebolavirus entry and will test our model by a combination of tried-and-true methods (X-ray crystallography, mutagenesis, and attachment and infectivity assays on viral pseudotypes and ebolaviruses alike) as well as innovative functional and biophysical approaches (artificial ebolaviruses, Deuterium Exchange Mass Spectrometry and Small Angle X-ray Scattering in solution) in order to address the following specific aims: (1) which residues comprise the RBS?; (2) to what extent does the mucin-like domain block the RBS?; (3) which residues are revealed by cathepsin cleavage?; and (4) is cathepsin cleavage the trigger of GP2 conformational change? This work represents the first structure-directed study of ebolavirus GP and will clearly map contents, conformation(s) and exposure of the receptor binding site. Results obtained by this work will elucidate mechanism(s) of ebolaviral entry, direct the search for the receptor(s), and provide templates for development and improvement of vaccines and therapeutics. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand if, and explain how the receptor binding site on ebolavirus becomes activated upon proteolytic cleavage during viral entry. This work will help us understand key vulnerabilities of the virus and will thus provide templates for development and improvement of vaccines and therapeutics.